Many gene families appear to have expanded through mechanisms such as unequal crossing over

n implicated in a variety of actin-mediated processes, including cell polarization, phagocytosis, chemotaxis, and morphogenesis. We, thus, examined the cytoskeletal organization of F-actin in wild-type and mutant cell lines, using the specific binding component rhodamineconjugated phalloidin. In wild-type cells, F-actin is primarily observed as a cortical band at the cell periphery with only diffuse cytoplasmic staining. limF- and chlimnulls exhibited nearly indistinguishable distributions PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19803731 of F-actin. However, overexpression of LimF or ChLim altered normal F-actin patterns. Both LimFOE and ChLimOE cells show a modest but consistent increase in F-actin-rich filopodia. Cells expressing the constitutively active Rab21Q66L showed enhanced ruffling of the cell surface, characterized by the prominent display of F-actin-rich blebs. Expression of the dominant-negative Rab21T21N also alters the organization of cortical F-actin, but in a quite different manner. Rab21T21N-expressing cells have extremely pronounced actin-rich `microspikes’ and filopodia-like structures. Mutation of the Rab21, LimF, or ChLim genes did not seem to correlate with many cellular or developmental changes. Protein overexpression or deficiency did not alter patterns of random movement or directed motility to either folate or cAMP or of growth in liquid, axenic media, although we did observe a mild cytokinesis defect for chlimnull cells; B5% of chlim-nulls had 44 nuclei during growth in shaking culture. In AZD-0530 web addition, all cell lines have substantially normal patterns of development under submerged conditions or on solid matrices. Cellular adhesive properties to a plastic substrate were similarly unchanged. On average, identical shear forces were required to disrupt cellsubstrate interactions among the different cell lines, although the ChLimOE cells may be slightly less adhesive. This contrasts the major adhesion defects in cells carrying deficiencies of other genes that regulate organization of the cell surface. Rab21, LimF, or ChLim also does not appear to have a significant function for fluid-phase uptake. We did, however, notice differences in the ability of the various cell lines to utilize bacteria as a food source. In the most striking phenotype, ChLimOE cells consistently have smaller plaques on bacterial lawns than wild-type controls, a phenotype that may be associated with a reduced ability to use bacteria as a nutrient source; reciprocally, chlim-null cells have expanded growth zones when grown on bacteria. LimF, ChLim, and Rab21-GTP cooperatively regulate phagocytosis through specific activating and inhibitory functions The altered growth patterns on bacterial lawns suggested an altered ability of the various cell lines to utilize bacteria as % cells attached at 65 r.p.m. 50 50 50 55 35 45 50 o5a o5a o10a o5a o5a Log-phase cells were plated in plastic culture dishes and shaken at varying speeds for 60 min at room temperature. Unattached cells were counted at each speed and normalized to the input cell number. A total of 50% of wild-type cells remained attached after shaking at 65 r.p.m. For the other cell lines, numbers listed reflect the percentage of cells attached under identical shaking conditions. a Data for the sadA, phg1, phg2, myoVII, and talin cell lines are extrapolated from previously published studies in a similar comparison with wild type. Rab21 regulation of phagocytosis T Khurana et al a nutrient source. This prompted us to investigate directly diff